Light activated gene transduction using long wavelength ultraviolet light for cell targeted gene delivery

ABSTRACT

In accordance with the present invention, methods are provided for treating a patient through the use of ultraviolet light activated gene therapy. Embodiments of the present invention include methods for the utilization of light activated gene therapy to repair and/or rebuild damaged cartilage by introducing a desired gene into a patient&#39;s tissue.

GOVERNMENT INTEREST

[0001] This invention was made with Government support under NIHContract #AR45972, an ROI grant awarded by NIAMS. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates generally to the field of gene therapy.According to the present invention, devices and methods are provided forthe combined use of light activated gene transduction (LAGT) employingultraviolet light and recombinant adeno-associated virus (r-AAV) for thepurpose of introducing a desired gene into a patient's tissue.

[0004] 2. Description of the Related Art

[0005] Somatic cell gene therapy is a form of treatment in which thegenetic material of a target cell is altered through the administrationof nucleic acid, typically in the form of DNA. In pursuit of effectivein vivo administration routes, scientists have harnessed the otherwisepotentially deleterious ability of viruses to invade a target cell and“reprogram” the cell through the insertion of viral DNA. Byencapsulating desirable genetic material in a viral particle, or“vector,” minus some of the viral DNA, the effective and targeteddelivery of genetic material in vivo is possible. As applied to specifictreatments, gene therapy offers the ability to adjust the expression ofdesirable molecules, including both intracellular and extracellularproteins, to bring about a desired biological result.

[0006] In particular, the desirable qualities of adeno-associatedviruses (AAV) have led to further study of potential gene therapy uses.As a vehicle for gene therapy recombinant forms of AAV, or r-AAV, offermany advantages including the vector's ability to infect non-dividingcells (e.g., chondrocytes, cells within cartilage), the sustained targetgene expression, the low immune response to the vector, and the abilityto transduce a large variety of tissues. The AAV contains a singlestrand DNA (ssDNA) genome. Under normal conditions AAV is present inhumans in a replication incompetent form, due to the fact the AAV alonedoes not encode the enzyme required for replication of the second DNAstrand. Successful r-AAV transduction often requires the presence of aco-infection with an adenovirus or the exposure of the host cell to DNAdamaging agents, such as γ-irradiation. The introduction of either theco-infection or the DNA damaging agents dramatically induces the ratelimiting step of second strand synthesis, i.e. the second strand of DNAwhich is synthesized based on the vector inserted first strand. However,making use of these DNA damaging agents is impractical because theadministration of an adenovirus co-infection to a patient is notpractical or desirable and the site specific and safety issues involvedwith using γ-irradiation undesirable as well.

[0007] In the past, attempts have been made to induce r-AAV transductionin vitro using UV radiation having a wavelength of 254 nm.Unfortunately, no effective therapeutic method or apparatus wasdeveloped based on these experiments due to the long exposure timesinvolved with using 254 nm UV radiation, the difficulties of delivering254 nm UV radiation to a surgical target site, and the inability toposition the 254 nm UV light source so as to allow effective penetrationof a target cell.

SUMMARY OF THE INVENTION

[0008] Preferred embodiments of the present invention provide structuresand methods for treating a patient using light activated gene therapy.

[0009] In accordance with an embodiment of the present invention, amethod of introducing a desired gene into a patient's tissue isprovided. The method includes locating a light probe proximate to thetarget cells. A long wavelength ultraviolet light is then transmittedthrough a light delivery cable to the light probe. Transduction of theultraviolet light activated viral vector in target cells is activatedusing the light probe. An ultraviolet light activated viral vector isdelivered proximate to target cells.

[0010] In accordance with another embodiment of the present invention, agene therapy system for increasing the transduction of an ultravioletactivated viral vector is provided. The system includes a light sourcecapable of long wavelength ultraviolet light output and a light probeconfigured to access an appropriate treatment site. An optical deliverycable is also provide to transmit the ultraviolet light from the lightsource to the light probe. In addition, light channeling optics areincluded to channel the light output into the light delivery cable.

[0011] In accordance with yet another embodiment of the presentinvention, a long wavelength ultraviolet radiation treatment system forincreasing the transduction of an ultraviolet light activated viralvector in a patient's target cells is provided. The system includes apower source which powers a light source producing a long wavelengthultraviolet light beam. A timed shutter with a shutter control interfaceis included to selectively block the light beam. In addition, an opticalcoupler for channeling the light beam into a light delivery cable. Alight probe, which is operatively connected to the light delivery cable,is configured to selectively output the light beam. Furthermore, thelight probe is also configured to access the target cells.

[0012] In accordance with still another embodiment of the presentinvention, an implant system for introducing a desired gene into apatient's tissue is provided. The system includes an implant configuredto be inserted into a patient's tissue in a minimally intrusive surgicalprocedure and an ultraviolet activated viral vector which is integratedwith the implant.

[0013] A feature of certain preferred embodiments of this invention isthe avoidance of the problems involved with using UV and γ-irradiationthrough the use of locally administered, long wavelength UV (i.e.,greater than or equal to 255 nm) radiation in order to induce the targetcell to more effectively stimulate the transduction of a UV activatedviral vector, such as recombinant adeno-associated virus (r-AAV).

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is flowchart of a method of treating target cells in apatient's tissue by activating the transduction of a UV light activatedviral vector using a light probe, in accordance with an embodiment ofthe present invention.

[0015]FIG. 2A is a side view schematic of a component of a longwavelength UV radiation system, including a light source and userinterface.

[0016]FIG. 2B is a schematic of another component of the long wavelengthUV radiation system, including a light probe, which in conjunction withthe light source and user interface shown in FIG. 2A, forms the in vivolong wavelength UV radiation system, in accordance with anotherembodiment of the present invention.

[0017]FIG. 2C is perspective schematic of an external light probe which,in conjunction with the component having the light source and userinterface shown in FIG. 2A, forms the ex vivo long wavelength UVradiation system configured for external applications, in accordancewith an alternate embodiment of the present invention.

[0018]FIG. 3 is a schematic of an injecting device for introducing a UVactivated vector into a patient's tissue, in conjunction with the longwavelength UV radiation system, shown in FIGS. 2A and 2B.

[0019]FIG. 4 is a method of treating a patient's cartilage using a UVactivated viral vector and a long wavelength UV radiation system, inaccordance with yet another embodiment of the present invention.

[0020] FIGS. 5A-5D are perspective schematics of implants for use inconjunction with the long wavelength UV radiation systems and methodprovided herein, in accordance with another embodiment of the presentinvention.

[0021]FIG. 5E is a cross-section schematic of the expanded implant ofFIG. 6D, the expanded implant shown located between two vertebra.

[0022]FIG. 6 is a flowchart of a method of treating a patient's tissueusing a light activated viral vector and a solid platform.

[0023] FIGS. 7-10 are graphs of the results of the proof of principleexperiment of Example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0024] The term “AAV” refers to adeno-associated virus, while “r-AAV”refers to recombinant adeno-associated virus. Preferably, r-AAV includesonly the desired gene to be introduced into the patient's tissue and theflanking AAV inverted terminal repeats (ITRs) that serve as thepackaging signals.

[0025] “Ultraviolet radiation” and “ultraviolet light,” also known as“UV”, refer to the portions of the electromagnetic spectrum which havewavelengths shorter than visible light. The range of wavelengthsconsidered to be ultraviolet radiation, from about 4 nanometers to about400 nanometers, is further subdivided into three subgroups, UVA, UVB,and UVC. “UVA” is the portion of ultraviolet radiation which includeswavelengths from 320 nm up to and including 400 nm. “UVB” is the portionof ultraviolet radiation which includes wavelengths from 280 nm up toand including 320 nm. “UVC” is the portion of ultraviolet radiationhaving a wavelength less than 280 nm.

[0026] The term “long wavelength UV” refers to ultraviolet radiation orlight having a wavelength equal to or greater than 255 nm, but not morethan 400 nm.

[0027] A “viral vector” refers to a virus, or recombinant thereof,capable of encapsulating desirable genetic material and transferring andintegrating the desirable genetic material into a target cell, thusenabling the effective and targeted delivery of genetic material both exvivo and in vivo. A. “UV activated viral vector” “UV light activatedviral vector” is any virus, or recombinant thereof, whose replication isregulated by ultraviolet light. Recombinant adeno-associated virus(r-AAV) is included in the group of viruses labeled UV activated viralvectors. A “solid platform” is any structure designed to be insertedinto the body for the purpose of aiding the treatment of the target siteproximate to where the solid platform is inserted.

[0028] The term “LAGT” refers to light activated gene transduction,while “LAGT probe” or “light probe” or “long UV wavelength light probe”refers to the medical device which delivers long wavelength ultravioletlight to the target site and effectuates the transduction of the desiredgene carried by the vector.

[0029] With reference to FIG. 1, a method of treating a patient's tissueis shown. A light probe is located 100 proximate to target cells. Longwavelength ultraviolet (UV) light is then transmitted 110 through alight delivery cable to the light probe. The transduction of the viralvector is activated 120 by locally administering ultraviolet light tothe target cells using the light probe. Preferably, the wavelength ofthe UV light ranges from about 255 nm up to and including about 400 nm.A UV activated viral vector containing a desired gene is delivered 130proximate to target cells in a patient's tissue. In other preferredembodiments, the wavelength of the UV light ranges from about 280 toabout 330. More preferably, the locally administered UV radiation has awavelength from about 315 nm to about 355 nm, most preferably about 325nm. In an alternate embodiment the ultraviolet radiation has awavelength of about 4 nm to about 400 nm, while in another alternateembodiment the ultraviolet radiation has a wavelength of 290 nm.

[0030] It should be noted that the method of FIG. 1 may be performed inother preferred embodiments in a different order than the textuallyoutlined above. For example, in another preferred embodiment the vectoris delivered prior to locally administering the ultraviolet light.

[0031] FIGS. 2A-2C illustrate separate components of a UV radiationdelivery system, with FIG. 2A showing the UV light generator 10, userinterface system, and FIG. 2B and FIG. 2C showing in vivo and ex vivoversions, respectively, of the light probe 26, 42. Note the light probe26, 42 is operatively connected to the UV light generator 10 by thelight delivery cable 24.

[0032]FIG. 2A shows a UV radiation delivery system including a lightsource 12 with sufficient long wavelength UV output. In addition, lightchanneling optics, such as an optical coupler 14, transmit the lightfrom the light source 12 into a light delivery cable 24, such as anoptical fiber cable or bundle that transmits the light to an targetsite, for in vivo purposes, via a light probe 26 (FIG. 2B). A timedshutter 16 is located in the path of the light beam between the lightsource 12 and the optical coupler 14 in order to control the length oftime the patient is exposed to UV light via the light probe 26 (FIG.2B). The timed shutter 16 is operatively connected via connectors 22 toa shutter controller 18 and a shutter control interface 20. Note thatthe in vivo light probe 26 disclosed in FIG. 2A can, in alternateembodiments in which ex vivo treatment is desirable, be interchangedwith an ex vivo light probe shown in FIG. 2C.

[0033]FIG. 2B shows a light probe 26 as part of an in vivo UV radiationdelivery system for use with the light source and user interface, suchas those shown in FIG. 2. The light probe 26 is configured to locallyirradiate target cells, infected by a UV activated viral vector, withlong wavelength ultraviolet (UV) light. The light probe 26 is joined tothe light delivery cable by an optical connector 28. The light probe 26is configured to fiber-optically transmit an appropriate UV wavelengthlight, which originates from the light source 12, through a light guide30 to a light guide terminator 34, such as a microlens tip orcylindrical diffusing lens tip, in order to “activate” r-AAVtransduction in target cells. The light probe 26 is preferably bothshaped in the form of an arthroscope and interchangeable with lightprobes having a differing configurations. For example, the light probecan be configured to have different forms in order to more effectivelyaccess different treatment sites. Preferably, the optical connector 28allows the light probe 26 to be selectively detached from the lightdelivery cable 24 when desired. In certain alternate embodiments, the UVradiation delivery system also includes a targeting laser beam (notshown) to enable accurate delivery of the light. Standard surgery toolsas recognized by those skilled in the art, for example cannulas andtrochars, may also be incorporated into the disclosed method.Preferably, the light probe 26 is configured to be sterile anddisposable.

[0034] In the embodiment shown in FIG. 2A, the light source 12 iscontained within a housing, while in certain alternate embodiments thelight source 12 is operatively joined to the housing. It should beunderstood that the exact shape and size of the light probe 26 shown inFIG. 2A, and especially the light probe tip, will vary depending on theparticular application and target site as would be understood by oneskilled in the art. For example, the light probe 26 can be configured toaccess an intervertebral disc in a patient's spine or the cartilage in apatient's joint. The preferred embodiments include a light sourcecomprising a laser tuned to the appropriate long UV wavelength. Inpreferred embodiments, the UV radiation delivery system, whether it be alamp or laser based system, will be optimized based on considerationssuch as cost and technical simplicity. In addition, the lamp deliverysystem can also include a targeting laser beam to enable accuratedelivery of the light. Standard surgery tools, for example cannulas andtrochars, may also be used.

[0035] As shown in FIG. 2C, in accordance with alternate preferredembodiments, an ex vivo light probe 42 for use with the light source anduser interface component of FIG. 2A is provided to form an ex vivo UVradiation system. In this ex vivo embodiment, the light probe 42 isdesigned for non-surgical use, such as the irradiation of a patient'sskin or irradiating tissue which has been removed from a patient for thepurpose of later being returned into the patient. The ex vivo configuredlight probe 42 has a handle 44, preferably a form fitting handleconfigured to allow the effective manual manipulation of the probe 42.The light probe 42 configured for external applications also has a shafthousing 46 surrounding a light guide 30 and a light guide terminator 34.An optical coupler 28 channels the light from the light delivery cable24 and preferably allows the light probe 42 to be selectively detachedfrom the light delivery cable 24 when desired.

[0036] Alternate embodiments employ as a light source, a lamp, such as ahigh intensity argon lamp. In these alternate embodiments, the UVradiation delivery system further includes a wavelength selectingdevice, such as a dichroic mirror and/or optical filter, set to transmitlong wavelength UV and reject unwanted light wavelengths. In theseembodiment, the wavelength selecting device and the dichroic mirror arepreferably contained in the same housing as the light source.

[0037] As shown in FIG. 3, an injecting device 36 having a housing 38and a plunger mechanism 40 is preferably employed in conjunction withthe UV radiation delivery system of FIGS. 2 and 2B. Preferably, theinjecting device 36 is configured for delivering a UV activated viralvector, such as r-AAV, to the target site using minimally invasivesurgical techniques. In alternate preferred embodiments, the injectingdevice can be configured to inject an implant or solid platform to atarget site in a patient (FIG. 6).

[0038] Surgery tools, other than injecting device shown in FIG. 3, whichcan be involved in certain preferred embodiments include a cannula, atrochar and other tools which the skilled artisan would recognize asbeing advantageous in conjunction with the embodiments provided herein.

[0039] Referring to FIG. 4, a method is provided for the treatment ofdamaged cartilage tear. A UV probe is inserted 200 proximate to acartilage target site. Preferably, if desirable, torn cartilage isremoved via standard arthroscopy. Long wavelength ultraviolet light(i.e., greater than or equal to 255 nm) is transmitted 210 to the targetcells via the fiber optic cable of the UV probe and the target cells areirradiated 220 with the long wavelength ultraviolet light in order toeffectuate the resurfacing of the target cartilage site. A UV activatedviral vector, such as r-AAV, is delivered 230 proximate to the targetsite, preferably by injection. It should be noted that the method ofFIG. 4 may be performed in other preferred embodiments in a differentorder than the textually outlined above.

[0040] Referring to FIGS. 5A-5E, alternate preferred embodiments providean implant system and methods for use thereof including the use ofimplants which serve as solid platforms at the target site (e.g., tocreate temporary mechanical rigidity between vertebra) while the targetcells respond to the introduction of the desired gene into the patient'stissue. Preferably, these carefully engineered implants can beexpandable in order to allow insertion through a minimal incision. Inaddition, these implants can be formed in a number of shapes, including(but not limited to) an unfolding geodosic dome 42 or tetrahedron (notshown), umbrella/dome (not shown), an expanding cylinder 44, and springswhich uncoil to increase diameter. Expanding cylinder 44 is shown in acompacted shape in FIG. 5A and an expanded state in FIG. 5B (and alsoFIG. 5E), while unfolding geodosic dome 42 is shown in a compacted shapein FIG. 5C and an expanded state in FIG. 5D. Preferably, these implantsare produced with implant integrated UV activated viral vector. Forexample, r-AAV can be integrated with the implant through bonding orcoating the r-AAV to the implant, absorbing the r-AAV into the implant,and/or baking the r-AAV to the implant surface. In alternate preferredembodiments the implant is delivered to a target site separate from theUV activated viral vector.

[0041]FIG. 5E shows a solid platform 44, to which a UV activated viralvector is preferably integrated, placed between two vertebra 50 in orderto facilitate the rebuilding or repair of the intervertebral disc 48.These solid platforms are preferably designed as surgical implants.Non-limiting examples of solid platforms with which UV activated viralvectors could be integrated include spinal spacers, as shown in FIG. 5E,and also total joint replacements such as hip implants, coronary stintsand other surgical implants. These examples are provided only forillustrative purposes and should not be considered in any way to limitthe present invention. Certain preferred embodiments of the presentinvention include a UV activated viral vector integrated with a solidplatform designed to facilitate the infection of cells proximate to thetarget site at which the solid platform is inserted. In an alternativeembodiment, the vector is delivered to the target site in a stepseparate from the insertion of the implant.

[0042] It should be understood that structural support implantsincorporating such conventional structures as, for example, but notlimited to, plates, rods, wire, cables, hooks, screws, are alsoadvantageously useful with preferred embodiments provided herein. Thesupport structure may be formed from material such as, but not limitedto, metal, carbon-fiber, plastic, and/or reabsorbable material.

[0043]FIG. 6 provides a method of treating a patient using UV activatedviral vector in conjunction with a solid platform. A UV activated viralvector containing a desired gene is integrated 300 with a solidplatform. Preferably, the vector is integrated with the solid platformby bonded, baked, coated, and/or absorbing. The solid platform is theninserted 310 into a patient proximate to target cells in a patient'stissue. A light probe is located 320 proximate to the target cells andlong wavelength ultraviolet light, having a wavelength from 225 nm to400 nm, is transmitted through a light delivery cable, such as a fiberoptic cable or bundle, to the light probe 330. The transduction of theviral vector is activated 340 by irradiating the target cells using thelight probe.

[0044] It should be noted that the method of FIG. 6, and the othermethods provided herein, may depending on the desired order and outcome,be performed in other preferred embodiments in a different order thanthe textually outlined herein.

[0045] Embodiments of the present invention include both in vivo and exvivo applications. In the ex vivo application the long wavelength UVlight dose is applied to cells or biological material external to thepatient and then delivered, preferably through injection, to the desiredsite of treatment. In the in vivo application the LAGT probe and the UVactivated viral vector are preferably introduced to the treatment siteusing minimally invasive surgical techniques, such as stab incisions.Alternate in vivo embodiments employ direct visualization surgicaltechniques.

[0046] A UV activated viral vector is any virus, or recombinant thereof,whose replication is regulated by ultraviolet light. Preferredembodiments of UV activated viral vectors are viruses with singlestranded DNA, the virus being capable of allowing a therapeuticallysignificant increase in virus transduction when a virus infected targetcell is exposed to a therapeutic does of ultraviolet radiation. Morepreferred embodiments include UV activated viral vectors capable ofinfecting non-dividing cells, effectuating sustained target geneexpression, eliciting a low immune response to the vector, andpossessing an ability to transduce a large variety of tissues.

[0047] Proof of principle experiments, both ex vivo and in vivo based,are currently under way and can determine the optimal wavelengths foractivating the gene therapy. The determination of more preferredwavelengths is based on among other factors, the ability to effectivelypenetrate a target cell, ease and efficiency of fiber optictransmission, the ability to trigger r-AAV transduction, and the lengthof time a patient must be exposed to receive a therapeutic dose ofultraviolet radiation. Preferably, the LAGT system delivers longwavelength ultraviolet radiation in the range of 315 nm to 400 nm.Current experiments support the use of ultraviolet radiation having awavelength from 315 nm to 355 nm, more particularly about 325 nm, but itis believed that these experiments will ultimately support ultravioletradiation having a wavelength from 315 nm to 400 nm. In addition,alternate embodiments employ a laser which produces ultravioletradiation having a wavelength of about 290 nm. Once specific wavelengthsare determined, the disclosed components can be optimized for thesespecific wavelengths.

[0048] The wavelength of the ultraviolet light generated in order toactivate UV activated viral vector transduction, including r-AAVtransduction, in target cells is preferably 255, 256, 258, 265, 275,285, 290, 295, 305, 314, 325, 335, 345, 355, 365, 375, 385, 395, or 400nanometers. More preferably, the wavelength of the ultraviolet light is290, 295, 300, 305, 310, 315, 316, 317, 322, 325, 327, 332, 337, 342,347, 352, 357, 362, 367, 372, 377, 382, 387, 392, 393, 394, 395, 396,397, 398, or 399 nanometers. Most preferably, the wavelength of theultraviolet light is 325 nanometers.

[0049] Tables 1-3 are charts of example growth factors, signalingmolecules and/or transcription factors which desired genes, selectedbased on the desired use (e.g., implant integrated vs. in solution) andoutcome (e.g., osteo-integration, spine fusion, perioprostheticosteolysis, and/or cartilage repair/regeneration) inserted into a UVactivated viral vector could encode for. The lists contained in Tables1-3 are provided for illustrative purposes and should not be taken aslimiting the embodiments of the invention in any way. TABLE 1osteo-integration and/or spine fusion: (a) GROWTH FACTORS TransformingGrowth Factor beta (TGFb) 1, 2 and 3 bone morphogenetic protein (BMP) 1,2, 4, 6 and 7 parathyroid hormone (PTH) parathyroid hormone relatedpeptide (PTHrP) fibroblast growth factor (FGF) 1, 2 insulin-like growthfactor (IGF) (b) SIGNALING MOLECULES AND TRANSCRIPTION FACTORS LMP-1Smad 1, 5, 8 dominant-negative Smad 2, 3 Smurf2 Sox-9 CBFA-1 ATF2

[0050] TABLE 2 perioprosthetic osteolysis: soluble tumor necrosis factorreceptors TNFR, TNFR:Fc osteoprotegerin (OPG) interleukin-1 receptorantagonist (IL-1RA), IL-1RII:Fc interleukin-4, 10 and viral IL-10

[0051] TABLE 3 12/21 LAGT for cartilage: (a) GROWTH FACTORS TransformingGrowth Factor beta (TGFb) 1, 2 and 3 bone morphogenetic protein (BMP) 1,2, 4, 6 and 7 parathyroid hormone (PTH) parathyroid hormone relatedpeptide (PTHrP) fibroblast growth factor (FGF) 1, 2 insulin-like growthfactor (IGF) osteoprotegerin (OPG) (b) SIGNALING MOLECULES ANDTRANSCRIPTION FACTORS Sox9 Smad 2, 3, dominant-negative Smad 1, 5, 8Smurf 1, 2 ATF2 CREB

[0052] The results of a completed proof of principle experiment areshown below in Example 1.

EXAMPLE 1

[0053] I. Methods

[0054] A. Isolation of Human Mesenchymal Stem Cells

[0055] Human Mesenchymal Stem Cells (hMSC) were isolated from patientblood samples harvested from the iliac crest. The blood samples werediluted in an equal volume of sterile Phosphate Buffered Saline (PBS).The diluted sample was then gently layered over 10 ml of Lymphoprep(Media Prep) in a 50 ml conical tube (Corning). The samples were thencentrifuged at 1800 rpm for 30 minutes. This isolation protocol is astandard laboratory technique, and the resulting gradient that formedenabled the isolation of the hMSCs from the layer immediately above theLymphoprep. The isolated fraction was placed into a new 50 ml conicaltube, along with an additional 20 ml of sterile PBS. The sample wascentrifuged at 1400 rpm for 8 minutes. The supernatant was removed thecell pellet was resuspended in 20 ml for fresh PBS, and centrifugedagain for 8 minutes at 1400 rpm. Afterwards the supernatant was removed,the cell pellet was resuspended in 10 ml of Dulbecco's Modified EagleMedium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1%Penicillin/Streptomycin (P/S) (Invitrogen). The hMSCs were grown andpassed as necessary in a 37°/5% CO₂, water-jacketed incubator (FormaScientific).

[0056] B. 325 nm UV treatment of Human Mesenchymal Stem Cells

[0057] Prior to irradiation, hMSCs were plated at a density of 5×10⁴cells/well in 12-well plates. The cells were allowed to sit downovernight. The next morning the media was removed immediately prior toirradiation. The cells were irradiated at various doses (500 J/m², 1000J/m², 3000 J/m², 6000 J/m², or 10,000 J/m²) of 325 nm UV light using ahelium-cadmium laser system (Melles Griot). After irradiation, freshmedia, either with or without recombinant adeno-associated virus wasadded to the wells.

[0058] C. Infection of Human Mesenchymal Stem Cells with RecombinantAdeno-Associated Virus

[0059] Infections were carried out in 12-well dishes. The cells wereinfected at various multiplicities of infection (MOIs=10, 100, and1000), using a recombinant adeno-associated virus carrying the bacterialβ-galactosidase reporter gene (rAAV-LacZ via UNC-Chapel Hill GeneTherapy Vector Core Facility). After being irradiated, the cells wereinfected with the predetermined amount of virus in a total volume of 500μl of DMEM/10% FBS/1% P/S. Two hours after the initial infection, andadditional 1 ml of media was added to the cultures. The cultures werethen allowed to incubate (37°/5% CO₂) for forty-eight hours beforeharvest for analysis.

[0060] D. Quantifying Recombinant Gene Expression

[0061] Forty-eight hours after infection, the cells were harvested; celllysates were made and analyzed using a commercially availableLuminescent β-gal Reporter System. (BD Biosciences), Briefly,experimental cell samples were removed from the 12-well dish using 0.25%Trypsin-EDTA. The cell suspension was transferred to a 1.5 ml conicaltube and the cells were pelleted via a 15 second centrifugation at13,000 rpm. The cell pellet was washed using two successive rounds ofresuspension in ice cold PBS and pelleting for 15 seconds at 13,000 rpm.The final pellet was resuspended in 75 μl of Lysis Buffer (100 mMK₂HPO₄, 100 mM KH₂PO₄, 1 M DTT) and subjected to three rounds offreeze/thaw in an isopropanol dry ice bath and a 37° water bath. Thelysates were centrifuged for a final time for 5 minutes at 13,000 rpm.Aliquots (15 μl) of the resulting supernatant were incubated with theprovided substrate/buffer solution for one hour and then analyzed usinga standard tube luminometer. The read out of this analysis is expressedin Relative Light Units (RLU) in the Results section.

[0062] II. Results

[0063] A. Exposure to 325 nm UV Increased the Level of Reporter GeneExpression

[0064] Exposure to 325 nm UV prior to infection with rAAV-LacZ had adose dependent increase in LacZ reporter gene expression at each of theMOI's used. The controls for each experiment were as follows: Mock(cells alone, no treatment) and cells treated with each of the variousUV dosages (500 J/m², 1000 J/m², 3000 J/m², 6000 J/m², which had RLUlevels consistent with the Mock cultures (data not shown). Statisticalsignificance was calculated using the Student T-Test. The results areshown below in FIGS. 7-10.

[0065] Although this invention has been disclosed in the context ofcertain preferred embodiments and an Example, it will be understood bythose skilled in the art that the present invention extends beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses of the invention and obvious modifications thereof. It willbe appreciated, however, that no matter how detailed the foregoing mayappear in text, the invention may be practiced in many ways. Thus, it isintended that the scope of the present invention herein disclosed shouldnot be limited by the particular disclosed embodiments described above,but should be determined only by a fair reading of the claims thatfollow and any equivalents thereof.

We claim:
 1. A method of introducing a desired gene into a patient'stissue comprising: locating a light probe proximate to target cells;transmitting long wavelength ultraviolet light through a light deliverycable to the light probe; activating transduction of an ultravioletlight activated viral vector in the target cells using the light probe;and delivering the ultraviolet light activated viral vector proximate tothe target cells.
 2. The method according to claim 1, wherein the longwavelength ultraviolet light transmitted to the light probe has awavelength of about 325 nm.
 3. The method according to claim 1, whereintransmitting the light occurs before delivering the vector.
 4. Themethod according to claim 1, wherein transmitting the light occurs afterdelivering the vector.
 5. The method according to claim 1, wherein theviral vector is recombinant adeno-associated virus (r-AAV).
 6. Themethod according to claim 1, wherein delivering the ultraviolet lightactivated viral vector proximate to target cells comprises infecting thetarget cells with the ultraviolet light activated viral vector.
 7. Themethod according to claim 5, further comprising: removing the tissuefrom the patient's body before exposing the tissue to the recombinantadeno-associated virus; and returning the tissue to the patient's body.8. The method according to claim 5, further comprising: exposing thetissue to the recombinant adeno-associated virus without first removingthe tissue from the patient's body.
 9. The method according to claim 5,wherein the long wavelength ultraviolet light transmitted to the lightprobe has a wavelength from about 315 nm to about 400 nm.
 10. The methodaccording to claim 5, wherein the long wavelength ultraviolet lighttransmitted to the light probe has a wavelength from about 380 nm toabout 330 nm.
 11. The method according to claim 5, wherein the longwavelength ultraviolet light transmitted to the light probe has awavelength from 315 nm to 355 nm.
 12. The method according to claim 5,wherein the long wavelength ultraviolet light transmitted to the lightprobe has a wavelength of about 325 nm.
 13. The method according toclaim 5, wherein the long wavelength ultraviolet light transmitted tothe light probe has a wavelength of about 290 nm.
 14. The methodaccording to claim 5, wherein the long wavelength ultraviolet lighttransmitted to the light probe is UVA light.
 15. The method according toclaim 5, wherein the long wavelength ultraviolet light transmitted tothe light probe is UVB light.
 16. The method according to claim 5,wherein the light probe is designed for arthroscopic surgery.
 17. Themethod according to claim 16, further comprising the step of discretelyinserting the light probe through minimally invasive surgical techniquesinto a patient.
 18. The method according to claim 17, wherein the targetcells are cartilage cells.
 19. The method according to claim 18, furthercomprising resurfacing the patient's cartilage by cartilage regenerationthrough the introduction of the desired gene into the patient's tissue.20. The method according to claim 1, further comprising: spacing atarget site, where the target cells are located, with an implant; andinjecting the ultraviolet light activated viral vector.
 21. The methodof claim 20, wherein spacing comprises attaching a structural supportimplant to bone.
 22. The method according to claim 1, furthercomprising: bonding a ultraviolet activated viral vector to a solidplatform configured to be surgically inserted in a patient fortherapeutic purposes; and surgically inserting the solid platform into apatient.
 23. The method according to claim 22, wherein the ultravioletlight activated viral vector is recombinant adeno-associated virus(r-AAV).
 24. The method according to claim 23, further comprising thestep of discretely inserting a light probe proximate to the solidplatform through a minimally invasive surgical technique into a patient.25. The method according to claim 1, wherein the transduction of theviral vector is activated by locally administering to the target cellslong wavelength ultraviolet light generated by a laser.
 26. The methodaccording to claim 1, wherein the desired gene introduced into thepatient's tissue is selected to effectuate tissue regeneration.
 27. Themethod according to claim 1, wherein the desired gene introduced intothe patient's tissue is selected to effectuate tissue repair.
 28. Themethod according to claim 1, wherein the desired gene introduced intothe patient's tissue is selected to effectuate tissue fusion.
 29. A genetherapy system for increasing the transduction of an ultravioletactivated viral vector comprising: a light source capable of longwavelength ultraviolet light output; a light probe configured to accessan appropriate treatment site; an optical delivery cable configured totransmit the ultraviolet light from the light source to the light probe;and light channeling optics configured to channel the light output intothe light delivery cable.
 30. The gene therapy system according to claim29, wherein the ultraviolet light activated viral vector comprisesrecombinant adeno-associated virus (r-AAV).
 31. The gene therapy systemaccording to claim 29, wherein the light source comprises a laser turnedto a therapeutically appropriate wavelength.
 32. The gene therapy systemaccording to claim 31, wherein the light source is a helium cadmiumlaser.
 33. The gene therapy system according to claim 29, wherein thelight source is a lamp.
 34. The gene therapy system according to claim33, further comprising: a wavelength selecting device set to transmit atherapeutically appropriate wavelength of ultraviolet light; andfocusing optics.
 35. The gene therapy system according to claim 29,wherein the gene therapy system is configured for the purpose ofminimally invasive surgery.
 36. The gene therapy system according toclaim 29, wherein the appropriate treatment site which the light probeis configured to access is a treatment site external to a patient beingtreated.
 37. The gene therapy system according to claim 29, wherein thelight source outputs light having a wavelength from about 315 nm toabout 400 nm.
 38. The gene therapy system according to claim 37, whereinthe light source outputs light having a wavelength of about 325 nm. 39.The gene therapy system according to claim 37, wherein the light sourceoutputs light having a wavelength of about 290 nm.
 40. The gene therapysystem according to claim 37, wherein the gene therapy system isconfigured for the purpose of increasing the therapeutic efficacy of apatient's treatment through the insertion of a desired gene in a UVactivated viral vector designed to be introduced into a patient'stissue.
 41. The gene therapy system according to claim 29, furthercomprising: a solid platform which includes the ultraviolet lightactivated viral vector, and a solid platform delivery device configuredto place the solid implant at the treatment site.
 42. A long wavelengthultraviolet radiation treatment system for increasing the transductionof an ultraviolet light activated viral vector in a patient's targetcells comprising: a power source; a light source producing a longwavelength ultraviolet light beam, the light source being powered by thepower source; a timed shutter configured to selectively block the lightbeam; a shutter control interface; a light delivery cable; an opticalcoupler for channeling the light beam into the light delivery cable; anda light probe operatively connected to the light delivery cable, thelight probe being configured to selectively output the light beam, thelight probe being further configured to access the target cells.
 43. Thelong wavelength ultraviolet radiation treatment system of claim 42,wherein the ultraviolet light activated viral vector comprisesrecombinant adeno-associated virus (r-AAV) and the light sourcecomprises a laser.
 44. The long wavelength ultraviolet radiationtreatment system of claim 43, wherein the light source is configured tooutput light having a wavelength from about 280 nm to about 330 nm. 45.The long wavelength ultraviolet radiation treatment system of claim 43,wherein the laser is configured to output light having a wavelength ofabout 325 nm.
 46. An implant system for introducing a desired gene intoa patient's tissue comprising: an implant configured to be inserted intoa patient's tissue in a minimally intrusive surgical procedure; and anultraviolet activated viral vector integrated with the implant.
 47. Theimplant system of claim 46, wherein the ultraviolet activated viralvector is recombinant adeno-associated virus (r-AAV).
 48. The implantsystem of claim 47, wherein the implant is both expandable and has abaked on coating of r-AAV.
 49. A kit for introducing a desired gene intoa patient's tissue, the kit comprising: an ultraviolet light activatedviral vector, the vector being capable of delivering the gene to thetissue; a light source capable of producing an ultraviolet light beam oflight having a wavelength from about 255 nm to about 400 nm; and a lightprobe operatively connected to the light source, the light probe beingconfigured to deliver the light to the tissue.
 50. The kit according toclaim 49, wherein the ultraviolet light has a wavelength from about 4 nmto about 400 nm.
 51. The kit according to claim 49, wherein theultraviolet light has a wavelength from about 280 nm to about 330 nm.52. The kit according to claim 49, wherein the ultraviolet light has awavelength of about 290 nm.
 53. The kit according to claim 49, whereinthe ultraviolet light has a wavelength of about 325 nm.
 54. The kitaccording to claim 53, wherein the light source is a helium cadmiumlaser.
 55. The kit according to claim 49, wherein the ultraviolet lighthas a wavelength from about 315 nm to about 355 nm.
 56. The kitaccording to claim 49, wherein the ultraviolet light has a wavelengthfrom about 315 nm to about 400 nm.
 57. The kit according to claim 49,wherein the viral vector is recombinant adeno-associated virus (r-AAV).58. The kit according to claim 49, wherein the ultraviolet light is UVAlight.
 59. The kit according to claim 49, wherein the ultraviolet lightis UVB light.
 60. The kit according to claim 49, wherein the light probeis designed for arthroscopic surgery.
 61. The kit according to claim 60,further comprising a sheath for minimally-invasive surgery.
 62. The kitaccording to claim 49, further comprising: a spacer configured to besurgically inserted in a patient for therapeutic purposes.
 63. The kitaccording to claim 62, wherein the spacer is adapted to preserve spacewithin a joint.
 64. The kit according to claim 62, wherein the viralvector is integrated onto the spacer.
 65. The kit according to claim 49,wherein the light source is a laser.
 66. The kit according to claim 49,wherein the desired gene is selected to effectuate tissue regeneration.67. The kit according to claim 49, wherein the desired gene is selectedto effectuate tissue repair.
 68. The kit according to claim 49, whereinthe desired gene is selected to effectuate tissue fusion.
 69. A kitaccording to claim 49, further comprising: a light delivery cableconfigured to transmit the ultraviolet light from the light source tothe light probe; and light channeling optics configured to channel theultraviolet light beam into the light delivery cable.
 70. The kitaccording to claim 49, wherein the light source is a lamp.
 71. The kitaccording to claim 70, further comprising: a wavelength selecting deviceset to transmit a therapeutically appropriate wavelength of ultravioletlight; and focusing optics.
 72. The kit according to claim 69, whereinthe light probe is configured to access a cartilage treatment site. 73.The kit according to claim 49, wherein the treatment site for which thelight probe is configured to access is a treatment site external to apatient being treated.
 74. The kit according to claim 69, furthercomprising: a solid platform which includes the ultraviolet lightactivated viral vector, and a solid platform delivery device configuredto place the solid implant at the treatment site.
 75. The kit of claim69, further comprising: a power source powering the light source; atimed shutter configured to selectively block the light beam; a shuttercontrol interface; and an optical coupler for channeling the light beaminto the light delivery cable.